ABSTRACT
It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network - including alpha-tocopherol (alphaT) and alphaT transfer protein - in aging in vivo, alpha-tocopherol transfer protein knockout (alphaTTP(-/-)) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% alphaT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F(2)alpha, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of alphaTTP(-/-) mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in alphaTTP(-/-) mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the alphaTTP(-/-) mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels. In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that alphaT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.
Subject(s)
Aging/physiology , Biomarkers/blood , Carrier Proteins/physiology , Fatty Acids, Unsaturated/blood , Motor Activity/drug effects , Vitamin E Deficiency/physiopathology , alpha-Tocopherol/metabolism , Animals , Ascorbic Acid/blood , Brain Chemistry , Dinoprost/analogs & derivatives , Dinoprost/blood , Female , Lipid Peroxidation/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/physiology , Oxidative Stress , Specific Pathogen-Free Organisms , Stereoisomerism , Thiobarbituric Acid Reactive Substances/metabolism , Ubiquinone/bloodABSTRACT
Ample evidence supports the critical role of oxidized low-density lipoprotein (ox-LDL) in initiation and progression of atherosclerosis. Oxidation of LDL is a complex process involving several steps (processes) of reactions such as initiation and propagation. Both proteins and lipids in LDL undergo free radical-mediated oxidations leading to the formation of ox-LDL that plays a pivotal role in atherosclerosis. Antioxidants of various types (both aqueous and lipophilic) either arrest or retard the oxidation of LDL at various steps of the oxidation process (e.g., initiation or propagation). Certain lipophilic antioxidants act as the chain-terminating antioxidants leading to the inhibition of LDL oxidation. The current chapter describes the designing and efficacy of two novel lipophilic antioxidants (benzofuranol, BO-653 and aniline, BO-313) in inhibiting the LDL oxidation and atherogenesis in experimental animal model. Furthermore, the characteristics of an effective antioxidant to inhibit LDL oxidation and atherogenesis which dictates the designing of the antioxidant drug and its mechanism(s) of antiatherogenic action are discussed.
Subject(s)
Antioxidants , Atherosclerosis/drug therapy , Aniline Compounds/chemical synthesis , Aniline Compounds/chemistry , Aniline Compounds/therapeutic use , Animals , Antioxidants/chemical synthesis , Antioxidants/chemistry , Antioxidants/therapeutic use , Benzofurans/chemical synthesis , Benzofurans/chemistry , Benzofurans/therapeutic use , Copper/chemistry , Disease Models, Animal , Drug Design , Female , Lipoproteins, LDL/metabolism , Male , Molecular Structure , Oxidation-Reduction , Rabbits , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
The pharmacological properties of MA-2029, a selective and competitive motilin receptor antagonist, were investigated in conscious dogs after oral administration. Gastrointestinal contractile activity was recorded by chronically implanted force transducers. The proximal gastric volume was measured with a barostat under constant pressure. Gastric emptying was examined using the paracetamol absorption test. MA-2029 (0.3-10 mg/kg, p.o.) administered in the interdigestive state inhibited gastrointestinal contractions induced by motilin (3 microg/kg, i.v.) in a dose-dependent manner. MA-2029 (0.3-3 mg/kg, p.o.) also inhibited the occurrence of spontaneous phase III contractions, even though MA-2029 had no effect on basal gastrointestinal motility or basal gastric emptying even at 10 and 30 mg/kg p.o. The inhibitory effect of MA-2029 on motilin-induced gastrointestinal motility corresponded to its plasma concentration. Motilin (0.3 microg/kg/h, i.v. infusion) reduced the proximal gastric volume by about 50% of control during isobaric distension. This effect was also inhibited by MA-2029 (1-10 mg/kg, p.o.) in a dose-dependent manner. In the digestive state, injection of motilin (3 microg/kg, i.v.) induced diarrhea in 9 of 11 dogs. MA-2029 (1-30 mg/kg, p.o.) reduced the incidence of diarrhea induced by motilin in a dose-dependent manner. The results indicate that MA-2029 inhibits hypermotility induced by motilin in conscious dogs without having an effect on the basal gastrointestinal tone or gastric emptying rate. MA-2029 may be useful in treating gastrointestinal disorders in which the pathogenesis involves the elevation of circulating motilin.
Subject(s)
Diarrhea/prevention & control , Gastric Fundus/drug effects , Gastrointestinal Motility/drug effects , Muscle Tonus/drug effects , Oligopeptides/pharmacology , Receptors, Gastrointestinal Hormone/antagonists & inhibitors , Receptors, Neuropeptide/antagonists & inhibitors , Administration, Oral , Animals , Diarrhea/chemically induced , Dogs , Dose-Response Relationship, Drug , Female , Gastric Emptying/drug effects , Gastric Fundus/physiology , Gastrointestinal Agents/administration & dosage , Gastrointestinal Agents/pharmacology , Gastrointestinal Motility/physiology , Male , Motilin , Muscle Contraction/drug effects , Muscle Tonus/physiology , Muscle, Smooth/drug effects , Oligopeptides/administration & dosageABSTRACT
Mitemcinal (GM-611) is a macrolide motilin receptor agonist with acid-resistance and without antibiotic activity. Since ABT-229 (a first generation of motilin receptor agonist) had failed to demonstrate symptomatic relief in functional dyspepsia and diabetic gastroparesis, there is a controversy for which of prokinetics or relaxants is clinically beneficial. Currently, oral mitemcinal has been focused on diabetic gastroparesis under clinical development. It showed to accelerate gastric-emptying in diabetic animals and in patients with gastroparesis. The latest double-blind, placebo-controlled study demonstrated to be effective at improving diabetes-related gastroparesis symptoms. A sub-group analysis, which included patients with BMI < 35 kg/m2 and hemoglobin A1c < 10%, there were significantly more symptomatic relieves in the 10 mg mitemcinal group than in the placebo group. The frequency of adverse events did not differ between groups. Mitemcinal shows promise in the subset of patients who should be confirmed in future studies.
Subject(s)
Diabetes Complications/complications , Diabetes Complications/drug therapy , Erythromycin/analogs & derivatives , Gastrointestinal Agents/therapeutic use , Gastroparesis/drug therapy , Gastroparesis/etiology , Animals , Diabetes Complications/pathology , Dogs , Erythromycin/chemistry , Erythromycin/therapeutic use , Gastric Emptying/drug effects , Gastrointestinal Agents/chemistry , Gastroparesis/pathology , Humans , Molecular Structure , Randomized Controlled Trials as TopicABSTRACT
To assess the antioxidative role of vitamin E (VE) in a mouse model of severe VE deficiency by using biomarkers, alpha-tocopherol transfer protein (alpha-TTP(-/-))-knockout mice were maintained on a VE-deficient diet for 28 weeks [KO group, n = 6]. Wild-type C57BL/6 mice were maintained on a diet containing 0.002% alpha-tocopherol [WT group, n = 6]. The animals were housed individually in a metabolic cage from the age of 9 weeks (Week 0) to 27 weeks. Urine was collected every week, and the levels of total hydroxyoctadecadienoic acid (tHODE), 7-hydroxycholesterol (t7-OHCh), and 8-iso-prostaglandin F(2alpha)(t8-isoPGF(2alpha)), which are biomarkers for lipid peroxidation, were measured by gas chromatography (GC)-mass spectrometry. From the age of 21 weeks (Week 12), three mice in each group were provided drinking water containing the water-soluble radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) until the end of the study (Week 19). Blood and tissue samples were collected, and the levels of the abovementioned biomarkers therein were assessed. AIPH consumption clearly elevated the plasma and erythrocyte levels of tHODE and t8-isoPGF(2alpha) in both the WT and KO groups except for the erythrocyte level of tHODE in the WT group. Furthermore, this elevation was more prominent in the KO group than in the WT group. Interestingly, AIPH consumption reduced the stereoisomer ratio of HODE (ZE/EE), which is reflective of the efficacy of a compound as an antioxidant in vivo; this suggests that free radical-mediated oxidation reduces the antioxidant capacity in vivo. The urine levels of tHODE, t7-OHCh, and t8-isoPGF(2alpha) tended to increase with AIPH consumption, but these individual levels fluctuated. It was clearly demonstrated by the proposed biomarkers that maintaining alpha-TTP(-/-) mice on a VE-deficient diet results in a severe VE deficiency and promotes lipid peroxidation.
Subject(s)
Azo Compounds/administration & dosage , Carrier Proteins/genetics , Diet , Imidazoles/administration & dosage , Lipid Peroxidation , Vitamin E Deficiency/metabolism , Water Supply , Animals , Azo Compounds/blood , Biomarkers/blood , Biomarkers/urine , Carrier Proteins/metabolism , Dinoprost/analogs & derivatives , Dinoprost/blood , Dinoprost/urine , Disease Models, Animal , Erythrocytes/chemistry , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/urine , Free Radicals/administration & dosage , Free Radicals/blood , Hydroxycholesterols/urine , Imidazoles/blood , Lipid Peroxidation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Time Factors , Tissue Distribution , Vitamin E Deficiency/genetics , alpha-Tocopherol/administration & dosageABSTRACT
The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.
Subject(s)
Motilin/drug effects , Muscle Contraction/drug effects , Oligopeptides/pharmacology , Receptors, Gastrointestinal Hormone/antagonists & inhibitors , Receptors, Neuropeptide/antagonists & inhibitors , Administration, Oral , Animals , Binding, Competitive , Cell Line , Colon/drug effects , Colon/metabolism , Dose-Response Relationship, Drug , Duodenum/drug effects , Duodenum/metabolism , Gastrointestinal Motility/drug effects , Humans , Male , Motilin/metabolism , Oligopeptides/administration & dosage , Oligopeptides/pharmacokinetics , Pain/drug therapy , Pain/physiopathology , Pain Measurement , RabbitsABSTRACT
It has been hypothesized that oxidative modification of low density lipoprotein plays a key role in the pathogenesis of atherosclerosis. In order to elucidate the role of lipid oxidation and its inhibition in vivo, apolipoprotein E and alpha-tocopherol (alphaT) transfer protein double knockout (ApoE(-/-)alpha-TTP(-/-)) and apolipoprotein E knockout (ApoE(-/-)) mice fed with a vitamin E-depleted diet and a diet containing 0.002 wt.% alphaT, respectively, were used with or without the treatment of a synthetic antioxidant 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653, 0.2 wt.%). The lipid oxidation markers of total hydroxylinoleic acid (tHODE), 8-iso-prostaglandin F(2alpha), and 7-hydroxycholesterol (t7-OHCh) in the blood, liver, and brain were inclusively measured with or without an excessive cholesterol-feeding (Ch-diet). The tHODE levels were elevated by Ch-diet in the plasma and brain of ApoE(-/-)alpha-TTP(-/-) mice and in the liver of ApoE(-/-) mice without BO-653. The levels of t7-OHCh in the liver were also increased due to the Ch-diet, and the ratio of t7-OHCh to the parent compound cholesterol was reduced to the control levels by BO-653. In summary, it was demonstrated by biomarkers, tHODE and t7-OHCh, that the added BO-653 in their diets exerted antioxidative effects in vivo under the condition of reduced vitamin E.
Subject(s)
Benzofurans/pharmacology , Fatty Acids, Unsaturated/metabolism , Hydroxycholesterols/metabolism , alpha-Tocopherol/pharmacology , Animals , Apolipoproteins E/genetics , Brain/metabolism , Carrier Proteins/genetics , Erythrocytes/chemistry , Erythrocytes/metabolism , Fatty Acids, Unsaturated/blood , Hyperlipidemias , Hypolipidemic Agents/pharmacology , Lipid Peroxidation , Liver/chemistry , Liver/metabolism , Male , Mice , Mice, Inbred C57BLABSTRACT
PURPOSE: To assess the role of vitamin E (VE) in age-related changes in the retinal tissues by using a mouse model of severe VE deficiency. METHODS: Pups of alpha-tocopherol transfer protein null (a-TTP(-)(/)(-)) mice were fed a VE-deficient diet for 4 or 18 months (VE (-) group). Wild-type C57BL/6 mice were fed a 0.002% alpha-tocopherol-supplemented diet (VE (+) group). In various ocular tissues, the VE levels were measured by high-performance liquid chromatography; the fatty acid composition by gas chromatography (GC); and the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F(2)(alpha) levels, which are biomarkers for lipid peroxidation, by GC-mass spectrometry. The retinal structure was assessed by light, electron, and fluorescence microscopy. RESULTS: The alpha-tocopherol level in the retinas obtained from 4-month-old VE (-) animals was 71-fold lower than that in the retinas obtained from the VE (+) group. In addition, gamma-tocopherol was not detected; thus, the VE (-) group demonstrated a more severe VE deficiency than ever reported. In this group, the concentration of n-3 polyunsaturated fatty acids decreased (0.3- to 0.9-fold), whereas that of other classes of fatty acids was unchanged or increased. At 18 months of age, the number of the outer nuclear layer (ONL) nuclei was observed to be 17% lower in the VE (-) than in the VE (+) group (P < 0.05). Electron microscopy revealed larger amounts of matrix between the ONL nuclei indicating the Müller cell hypertrophy, greatly expanded rod outer segment discs, and a larger number of inclusion bodies in the retinal pigment epithelium (RPE; P < 0.05) in the VE (-) group. Fluorescence microscopy revealed that the autofluorescence signal was increased in the RPE layer in this group. When the observations of the 18-month-old animals were compared to those of the 4-month-old animals, the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F(2)(alpha) levels were found to be increased in the retina and RPE obtained from both the VE (-) and VE (+) groups; however, the age-related increases were more remarkable in the VE (-) group (2.6- to 43.5-fold) than in the VE (+) group (0.8- to 8.7-fold). CONCLUSIONS: The combined use of a-TTP(-)(/)(-) mice and a VE-deficient diet leads to a severe deficiency of VE, enhances lipid peroxidation in the retina, and accelerates degenerative damage of the retina with age.
Subject(s)
Carrier Proteins/genetics , Macular Degeneration/etiology , Vitamin E Deficiency/complications , Vitamin E/physiology , Animals , Chromatography, Gas , Chromatography, High Pressure Liquid , Diet , Dinoprost/analogs & derivatives , Dinoprost/metabolism , Fatty Acids/metabolism , Fatty Acids, Unsaturated/metabolism , Gene Silencing/physiology , Lipid Peroxidation , Macular Degeneration/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Pigment Epithelium of Eye/metabolism , Pigment Epithelium of Eye/ultrastructure , Retina/metabolism , Retina/ultrastructure , Vitamin E/analogs & derivatives , Vitamin E/metabolism , Vitamin E Deficiency/pathologyABSTRACT
Vitamin E has failed to protect humans from cardiovascular disease outcome, yet its role in experimental atherosclerosis remains less clear. A previous study (Proc. Natl. Acad. Sci. USA 97:13830-13834; 2000) showed that vitamin E deficiency caused by disruption of the alpha-tocopherol transfer protein gene (Ttpa) is associated with a modest increase in atherosclerosis in apolipoprotein E gene deficient (Apoe(-/-)) mice. Here we confirm this finding and report that in Apoe(-/-)Ttpa(-/-) mice dietary alpha-tocopherol (alphaT) supplements restored circulating and aortic levels of alphaT, and decreased atherosclerosis in the aortic root to a level comparable to that seen in Apoe(-/-) mice. However, such dietary supplements did not decrease disease in Apoe(-/-) mice, whereas dietary supplements with a synthetic vitamin E analog (BO-653), either alone or in combination with alphaT, decreased atherosclerosis in Apoe(-/-) and in Apoe(-/-)Ttpa(-/-) mice. Differences in atherosclerosis were not associated with changes in the arterial concentrations of F(2)-isoprostanes and cholesterylester hydro(pero)xides, nor were they reflected in the resistance of plasma lipids to ex vivo oxidation. These results show that vitamin E at best has a modest effect on experimental atherosclerosis in hyperlipidemic mice, and only in situations of severe vitamin E deficiency and independent of lipid oxidation in the vessel wall.
Subject(s)
Atherosclerosis/genetics , Atherosclerosis/therapy , Dietary Supplements , Vitamin E/pharmacology , Animals , Apolipoproteins E/genetics , Atherosclerosis/metabolism , Benzofurans/pharmacology , Lipid Peroxidation , Lipoproteins/metabolism , Male , Mice , Mice, Transgenic , Oxidative Stress , Oxygen/metabolism , Ubiquinone/pharmacology , Vitamin E Deficiency , alpha-Tocopherol/metabolismABSTRACT
Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse has been widely used as a rodent model of Parkinson's disease. In this study, alpha-tocopherol (alphaT) transfer protein knockout (heteromutant type, alpha-TTP((+/-))) mice were used to evaluate the protective effects of alphaT and gamma-tocopherol (gammaT) against MPTP-induced neurotoxicity. The intraperitoneal administration of MPTP to mice induced a decrease in the striatal levels of dopamine (DA) 3 days after the administration in both alpha-TTP((+/-)) and wild-type mice; these mice were fed an alphaT-deficient diet for 3 weeks before the MPTP administration. The DA levels in the alpha-TTP((+/-)) mice, which had been fed a gammaT-fortified diet (0.10 wt.%) for 3 weeks and were administered with MPTP, were recovered to those of the control, whereas there was no significant protective effect of alphaT despite the considerably higher striatal concentration of alphaT than gammaT. The immunohistochemical study also revealed that gammaT exerted a protective effect against neurodegenerative toxicity of MPTP. Collectively, this is the first report showing that the protective effect of gammaT is stronger than that of alphaT against the MPTP-induced damage of dopaminergic neurons in the mouse.
Subject(s)
Antioxidants/pharmacology , Brain/drug effects , Dopamine/metabolism , Vitamins/pharmacology , alpha-Tocopherol/pharmacology , gamma-Tocopherol/pharmacology , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Animals , Brain/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/drug effects , Neurons/metabolismABSTRACT
OBJECTIVE: The relevance of oxidative stress in mice fed a choline-deficient diet (CDD) was investigated in relation to the oxidative stress marker, hydroxyoctadecadienoic acid (HODE) in comparison with F2-isoprostanes. Further, the protective effects of antioxidants against oxidative damage were assessed by using HODE. METHODS: We recently proposed total HODE as a biomarker for oxidative stress in vivo. Biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert various oxidation products of linoleates to HODE. In the present study, this method was applied to measure oxidative damage in mice induced by CDD for 1 mo. RESULTS: CDD, when compared with choline-controlled diet (CCD), increased liver weight and fatty acid accumulation but the increase in body weight was less significant. Remarkable increases in HODE and 8-iso-prostaglandin F(2alpha) in liver and plasma were observed when mice were fed with the CDD for 1 mo compared with the CCD. The HODE level was about two to three orders higher than the F2-isoprostane level. This increase was decreased to the level of the CCD when alpha-tocopherol or 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, a potent synthetic antioxidant, was mixed with the CDD. The stereoisomer ratio of HODE (9-and-13 (Z,E)-HODE/9-and-13 (E,E)-HODE) was decreased by CDD compared with CCD, which was spared by the addition of alpha-tocopherol and 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran. However, the increase in plasma glutamic-pyruvic transaminase and fatty acids in liver induced by the CDD was not recovered by any antioxidant. CONCLUSIONS: This study clearly demonstrated that oxidative stress was involved in fatty liver formation induced by the CDD and that HODE was a good biomarker for an oxidative stress in vivo.
Subject(s)
Choline Deficiency/metabolism , Dinoprost/analogs & derivatives , Fatty Acids, Unsaturated/metabolism , Fatty Liver/metabolism , Lipid Peroxidation , Animals , Antioxidants/analysis , Antioxidants/metabolism , Benzofurans , Biomarkers/analysis , Biomarkers/blood , Biomarkers/metabolism , Choline/administration & dosage , Choline/metabolism , Chromatography, High Pressure Liquid , Dinoprost/analysis , Dinoprost/blood , Dinoprost/metabolism , Fatty Acids, Unsaturated/analysis , Fatty Acids, Unsaturated/blood , Male , Mice , Mice, Inbred C57BL , Organ Size/drug effects , Oxidation-Reduction , Oxidative Stress , Random Allocation , Specific Pathogen-Free OrganismsABSTRACT
We have recently proposed total hydroxyoctadecadienoic acid (HODE) as a biomarker for oxidative stress in vivo. The biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert the oxidation products of linoleate to HODE. In the present study, this method was applied to measure the oxidative damage induced by the administration of carbon tetrachloride to mice and also to evaluate the capacity of antioxidant to inhibit the above damage. alpha-Tocopherol transfer protein knock out (alpha-TTP-/-) mice were used to evaluate antioxidant effect in the absence of alpha-tocopherol. The intraperitoneal administration of carbon tetrachloride to mice induced the increase in HODE in liver and plasma, which was followed by an increase in plasma glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). F2-isoprostanes, another prevailing biomarker, were also increased similarly, but their concentration was approximately two to three orders of magnitude smaller than that of HODE. The lipophilic antioxidants such as gamma-tocopherol, gamma-tocotrienol and 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) were effective in suppressing the formation of HODE.
Subject(s)
Antioxidants/pharmacology , Biomarkers/analysis , Carbon Tetrachloride/toxicity , Fatty Acids, Unsaturated/analysis , Lipid Peroxidation/drug effects , Alanine Transaminase/blood , Animals , Antioxidants/administration & dosage , Ascorbic Acid/blood , Aspartate Aminotransferases/blood , Benzofurans/antagonists & inhibitors , Benzofurans/blood , Benzofurans/pharmacology , Biomarkers/blood , Biomarkers/metabolism , Carbon Tetrachloride/administration & dosage , Carrier Proteins/genetics , Chromatography, High Pressure Liquid , Diet , Dinoprost/analogs & derivatives , Dinoprost/blood , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/metabolism , Genotype , Injections, Intraperitoneal , Lipid Peroxides/blood , Lipid Peroxides/urine , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress , Specific Pathogen-Free Organisms , Thiobarbituric Acid Reactive Substances/metabolism , Tocotrienols/antagonists & inhibitors , Tocotrienols/blood , Tocotrienols/pharmacology , Ubiquinone/analogs & derivatives , Ubiquinone/blood , Vitamin E/administration & dosage , Vitamin E/pharmacology , alpha-Tocopherol/bloodABSTRACT
4,6-Di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 muM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of alpha-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di-tert.-butyl-2,3,5,7a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.
Subject(s)
Antioxidants/pharmacology , Benzofurans/pharmacology , Lipid Metabolism , Lipid Peroxidation , Peroxides/metabolism , Albumins/metabolism , Ascorbic Acid/metabolism , Bilirubin/metabolism , Cholesterol Esters/metabolism , Humans , Oxidation-Reduction , Plasma/metabolism , Stereoisomerism , Uric Acid/metabolism , alpha-Tocopherol/metabolismABSTRACT
BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, is a synthetic antioxidant which is now being developed as an anti-atherogenic drug. The antioxidant action of BO-653 against lipid peroxidation in rat plasma was studied and compared with its analogue BO-653M, 2,3-dihydro-5-hydroxy-4,6-di-methyl-2,2-dipentylbenzofuran, and vitamin E. BO-653 was readily incorporated into plasma by oral administration and it inhibited plasma lipid peroxidation more efficiently than vitamin E independent of the presence or absence of vitamin C. On the other hand, its analogue BO-653M having two methyl substituents in place of tert-butyl groups of BO-653 did not inhibit the lipid peroxidation in plasma as efficiently as BO-653, demonstrating clearly that the tert-butyl groups at the ortho-position play a key role in determining the antioxidant efficacy.
Subject(s)
Antioxidants/pharmacology , Benzofurans/pharmacology , Lipid Peroxidation/drug effects , Animals , Arteriosclerosis/metabolism , Ascorbic Acid/pharmacology , Male , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Vitamin E/pharmacologyABSTRACT
Antioxidants have been considered as potential antiatherogenic agents by inhibiting oxidation of low-density lipoprotein (LDL), albeit vitamin E, a natural antioxidant, has failed to show reduction on atherosclerosis in clinical trials. We have rationally designed and synthesized a novel series of antioxidants, 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols, to overcome the clinical limitation of vitamin E. In vitro, the compounds showed a potent inhibitory effect on lipid peroxidation detected as 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA)-dependent chemiluminescence in linoleic acid autoxidation. They also inhibited the LDL oxidation induced by Cu(2+), and the inhibition is more potent than that of vitamin E and probucol. In vivo, 4,6-di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653, 1f), an optimal compound, showed the highest concentration in plasma and LDL fraction in Watanabe heritable hyperlipidemic rabbits, due to its high affinity to LDL. The isolated LDL samples from the 1f-treated rabbits showed potent resistibility to LDL oxidation. Compound 1f has been taken into clinical trials.
Subject(s)
Antioxidants/chemical synthesis , Arteriosclerosis/prevention & control , Benzofurans/chemical synthesis , Animals , Antioxidants/pharmacokinetics , Antioxidants/pharmacology , Benzofurans/pharmacokinetics , Benzofurans/pharmacology , Free Radicals , Linoleic Acid/chemistry , Lipoproteins, LDL/blood , Lipoproteins, LDL/chemistry , Luminescent Measurements , Oxidation-Reduction , Rabbits , Structure-Activity Relationship , alpha-Tocopherol/pharmacologyABSTRACT
Antioxidants have been proposed as a promising treatment for restenosis after percutaneous transluminal coronary angioplasty (PTCA), but their mechanism of action remains unclear. Here, we investigated the effect of antioxidants on gene expression in the artery after balloon denudation. We developed a sensitive ribonuclease (RNase) protection assay for the messenger RNA (mRNA) levels of immediate early (IE) genes (c-jun, c-fos and c-myc), as well as platelet-derived growth factor-A (PDGF-A), platelet-derived growth factor-beta receptor, transforming growth factor-beta 1, and vascular endothelial growth factor. New Zealand White rabbits were fed a 0.17% cholesterol diet containing vehicle, BO-653 or probucol, and balloon denudation for iliac arteries was performed. The iliac arteries were then removed at 4 h after the denudation, for IE genes, and 10 days after for growth factors and receptors. Both BO-653 and probucol significantly reduced neointimal thickening, compared with the control. In terms of gene expression, BO-653, but not probucol, significantly inhibited c-myc induction. On the other hand, probucol, but not BO-653, significantly inhibited PDGF-A expression. Neither treatment had any effect on the expression of other genes. These results suggest that antioxidants affect the gene expression of the neointimal response and that both BO-653 and probucol inhibit gene expression in specific manners.
Subject(s)
Benzofurans/pharmacology , Cholesterol, Dietary/administration & dosage , Genes, myc/drug effects , Iliac Artery/pathology , Platelet-Derived Growth Factor/drug effects , Probucol/pharmacology , RNA, Messenger/analysis , Analysis of Variance , Animals , Antioxidants/pharmacology , Arteriosclerosis/genetics , Arteriosclerosis/therapy , Base Sequence , Catheterization , Diet, Atherogenic , Disease Models, Animal , Gene Expression , Genes, myc/genetics , Male , Molecular Sequence Data , Platelet-Derived Growth Factor/genetics , Probability , Rabbits , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di-tert-butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of alpha-tocopherol on the efficacy of radical scavenging within the membranes. Di-tert-butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert-butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.